Cancer is a leading cause of death in the United States and worldwide. As such the President of the United States has recently established the White House Cancer Moonshot Task Force, the mission of which is to eliminate cancer as we know it. Part of this mission is to encourage development of novel cancer treatments. This innovative multi-PI proposal represents a novel approach to this challenge. Oncogenic activation of the Ras family of GTPases occurs in ~30% of cancers making it the most frequently mutated oncogene in human cancers. Despite a great deal of progress in our understanding of the biochemistry of Ras and it's role in tumorigenesis, development of effective therapeutic inhibitors of Ras to date has been disappointing. Thus, there remains a critical need to develop targeted inhibitors of this oncoprotein for treatment of patients with Ras-positive tumors. Using an unbiased, protein engineering approach, we will develop high affinity reagents that specifically target each of the three Ras isoforms present in human tumors, namely H-Ras, K-Ras, and N- Ras. These reagents will then be used to develop small molecule inhibitors to Ras. We propose 4 major aims to accomplish our goals. In Aim 1, we will utilize the monobody platform to isolate highly specific affinity reagents that target each Ras isoform and characterize their in vitro specificity. In Aim 2, we will determine the effect of these monobody reagents on Ras-mediated signaling activity, focusing primarily on the ERK-MAPK and PI3K-AKT pathways which are the major targets of oncogenically activated Ras. Aim 3 will then define the biologic activity of monobodies at inhibiting Ras-mediated transformation in several pre-clinical models including 2D/3D culture systems, mouse xenograft assays and transgenic animal models for Ras tumorigenesis. Finally, Aim 4 will utilize a high content screening approach to identify small molecule mimetics of these monobody inhibitors which will serve as lead Ras inhibitors. These studies represent a unique and powerful approach toward developing highly specific Ras inhibitors and thus have the potential to make a major impact on cancer therapy. In addition, this project is highly relevant to the mission of National Cancer Institute's recently established Ras Initiative at the Frederick National Laboratory for Cancer Research which is charged with targeting Ras-dependent cancers.